Project Summary Cilia serve as sensory devices on most eukaryotic cells surface and play an essential role in development. Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are highly conserved in all ciliated organisms. With rapid advancements in the positional cloning of human disease genes in the past decade, a wide variety of disorders such as autosomal dominant polycystic kidney disease (ADPKD) have been characterized molecularly as ciliopathies. Consistent with the ubiquitous presence of cilia, many ciliopathies occur as syndromic disorders that affect multiple organs, including the kidney, liver, limb, eye, and central nervous system. One central question in cilia biology is that how the ciliary gate functionally separates the cilium from the cell body and makes it a discrete sensing organelle. During ciliogenesis, the distal appendages of the mother centriole transform to transition fibers (TFs), which form a 9-bladed propeller structure connecting the basal body to the ciliary base membrane. The distinct subcellular location of TFs makes it a good candidate for the ciliary gate. Nonetheless, the paramount challenges being that molecular insights about the establishment, either structural or functional, of TFs as the ciliary gate remain poorly defined. Due to the essential roles of cilia in mammalian embryonic development, the study of the connections between cilia and disease are extremely difficult in mammalian models. Thus, alternative experimental systems are necessary. Caenorhabditis elegans has been established as an effective model for characterizing the physiological roles of ciliary proteins in their native cellular environments that is relevant for understanding mammalian biology due to the highly conserved cilia composition and signaling. We pioneered the application of C. elegans as a model to study the biological importance of TFs. Our preliminary studies show that DYF-19 physically associates with different players to regulate distinct cilia gating: with the DYF-19-TALPID-3-ANK-26 functional module in regulating IFT import, whereas DYF-19-CCDC-85 module in regulating gating for membrane proteins. On the other hand, HYLS1 coordinate with GAS8 to regulate the establishment of the ciliary gate. We also retrieved novel worm mutants with likely disrupted TF integrity in a forward genetic screening. Furthermore, our initial studies suggested that the key discoveries made in C. elegans are highly conserved in mammalian cells. In this proposal, we will determine the full components and activities of underlying pathways so that the fundamental roles of the ciliary gate in the context of cilia and ciliopathies are better understood. We plan to achieve this goal by pursuing three specific questions: i), how cilia gating is achieved? ii) how the ciliary gate is established? and iii) if the core pathways for the ciliary gate are conserved in mammalian cells? By combining C. elegans with mammalian systems, we are confident to provide seminal information about the molecular identity and the core conserved pathways of the ciliary gate, and substantially extend our understanding of cilia biology as well as of the pathogenesis of human ciliopathies.